The immune system of a mammalian organism produces antibodies which are also known as immunoglobulins as a response to the introduction of foreign substances. They serve as a defense against foreign substances which are also referred to as antigens. The immunoglobulins can be divided into five different classes. One distinguishes between immunoglobulins of the M, G, A, E and D classes. These five immunoglobulin classes differ in the composition of the heavy chain which is referred to as the μ, γ, α, ε and δ chain, respectively.
Each immunoglobulin class has a different function in the organism. The class M immunoglobulins occur during a first contact with the antigen, the so-called primary immunization. However, the concentration of these immunoglobulins rapidly decreases as the infection progresses. The class G immunoglobulins are firstly slowly formed during a primary immunization and occur in large amounts in a second infection with the same antigen. The class A immunoglobulins are found on the surfaces of the mucous membranes of the organism and are responsible for the local defense processes. The class E immunoglobulins are mainly responsible for allergic reactions. The exact function of the class D immunoglobulins is thus far unknown.
The individual immunoglobulin classes occur in very different concentrations in the blood. Thus, the class G immunoglobulins (IgG) are the most abundant class in normal human serum with a proportion of about 75% which corresponds to a serum content of 8 to 18 mg/ml. The second most frequent immunoglobulin is IgA, the average serum concentration of which is 0.9 to 4.5 mg/ml. Class M immunoglobulins are present at a concentration of 0.6 to 2.8 mg/ml, class D immunoglobulins are present at a concentration of 0.03 to 0.4 mg/ml. The proportion of IgE antibodies is lowest—they only occur in serum at a concentration of 0.02 to 0.05 μg/ml.
Various methods for detecting antibodies of a certain class that are specific for an antigen are described in the prior art. Thus, the detection of antigen-specific antibodies of a certain class in a sample is often carried out by binding specific antibodies to a solid phase coated with the specific antigen. The immunoglobulins (Ig), which are specific for the antigen and are now bound to the solid phase, are detected by the binding of antibodies, which are specifically directed against human Ig of a certain class, to the Ig molecules to be detected. The antibodies directed against human Ig are provided with a label by means of which the detection takes place. However, such a test procedure (indirect test format) is only possible if, before the reaction with the class-specific labelled antibodies directed against human Ig, all unspecific, non-bound Ig is removed by washing. A one-step test procedure such as that, which is often required for automated systems, is thus not possible in this format.
The so-called bridge test opens up a possibility for carrying out an antibody detection in a one-step test. The bridge test concept is described in EP-A-0 280 211. In this method, a first binding partner, which is capable of specific binding to the antibody to be determined such as, for example, an antigen, is bound to a solid phase. The antibody to be determined binds to the solid phase-bound antigen. A further specific antigen, which is provided with a label, is also present in the test mixture. The antibody is detected by means of the label. If immunoglobulins of different classes but of the same specificity are present in the sample, the test does not distinguish between the various classes.
EP 0 944 838 B1 discloses a bridge test detection method that enables the selective determination of antigen-specific IgG antibodies in a sample which also contains IgM antibodies of the same specificity. The antigen is used in a monomeric form for this. The monomeric antigen has the effect that IgM antibodies having the same specificity cannot react or can only weakly react with the antigen.
Specific antibody tests are of major importance in infection serology in which the immune response to antigenic structures of pathogens is detected. Although different sources for obtaining antigens of the pathogens are state of the art (recombinant expression in prokaryotic and eukaryotic systems, controlled culture of the pathogen, isolation of antigens from natural sources) it is not always possible to obtain antigens in monomeric form in order to carry out an IgG-selective immunoassay in a one-step format.
EP 0 957 360 B1 describes the use of rheumatoid factors to reduce the Hook effect in methods for determining an analyte.
EP 1 098 198 A1 describes an immunoassay for the determination of human IgG antibodies. In this case a particular monoclonal antibody is used, which is directed against a conformation epitope of the Fc fragment of human IgG bound to an antigen. This antibody is used for the selective detection of IgG and can differentiate between IgGs that bind to an antigen and IgGs that bind no antigen. However, the presence of unbound IgG interferes and leads to signal losses.
EP 1 653 233 A1 describes a test procedure for the determination of antigen-specific antibodies of the IgG class wherein a sample is brought into contact with a solid phase to which antigens are bound. In this method a monoclonal antibody is used that distinguishes between immunoglobulins unspecifically bound to the solid phase and immunoglobulins bound to the antigen. After contacting the sample with the solid phase, it is washed and, subsequently, the monoclonal antibody is applied.
An immunoassay for detecting antigen-specific immunoglobulin G in a one-step format that can be carried out using automated analyzers is described in the patent application WO 99/15898 (Chien et al.). In Chien et al, an antibody is used which is specific for the constant part of the human immunoglobulin G. In this assay, there is a significant interference by free unspecific IgG without sufficient discrimination between free and complexed IgG.
One-step methods of the prior art for the detection of IgG cannot adequately distinguish between free and bound IgG. In order to discriminate between IgG and IgM of the same specificity, antigens must be monomeric which is a requirement that cannot often be fulfilled.
Hence the object of the present invention was to develop a method for the detection of an analyte in a sample and, in particular, antigen-specific antibodies of the immunoglobulin class G that can be carried out in a one-step method in order to be used advantageously in automated systems. In addition, the present invention has the object of at least partially overcoming the disadvantages of the prior art. For this, it is necessary to adequately discriminate between free analyte and bound analyte.